Cardiovascular disease in patients with diabetes is a pressing clinical problem. The endothelium is vital to the maintenance of vascular homeostasis. Alteration of endothelial cell phenotype leading to loss of nitric oxide bioactivity and increased endothelial inflammatory activation via NF:B may contribute to atherogenesis and clinical vascular events in diabetes. Recent experimental studies indicate that insulin signaling through the PI3K-Akt pathway contributes to endothelial nitric oxide production. Abnormalities of these signaling mechanisms produce endothelial insulin resistance and impair vascular function in animal models. Activation of PKC2 may link the metabolic derangements of diabetes to endothelial insulin resistance and pro-inflammatory endothelial phenotype. Despite the potential clinical relevance of altered endothelial phenotype, few studies have translated these experimental findings to patients with diabetes. Our preliminary data show that endothelial inflammatory activation is associated with vascular dysfunction and diabetes using a novel method to collect and characterize endothelial cells from human subjects. We now propose to test the hypothesis that altered endothelial cell phenotype contributes to vascular dysfunction in patients with diabetes. In Aim 1, we will characterize the relation of endothelial cell insulin resistance and inflammatory activation to vascular dysfunction and diabetes mellitus in human subjects. We will relate markers of endothelial insulin signaling (eNOS expression, insulin-mediated eNOS phosphorylation) and evidence of endothelial inflammatory activation (I:2, p65, ICAM expression) in endothelial cells freshly isolated from human subjects to the presence of diabetes and to the severity of endothelial vasomotor dysfunction (flow-mediated dilation, peripheral arterial tonometry and reactive hyperemia) in 120 patients with diabetes and 120 control subjects. In Aim 2, we will investigate the contribution of PKC2 activation and NF:2 activation to adverse endothelial phenotype and impaired vascular function. We hypothesize that endothelial cell insulin resistance and inflammatory activation will be reversed with in vitro treatment with selective PKC2 inhibitor (LY379196) or an NF:2 inhibitor (sodium salicylate), supporting a role for inflammation and PKC2 activation in patients with diabetes. In Aim 3, we will gain evidence that altered endothelial cell phenotype contributes to impaired nitric oxide bioavailability in 40 patients with diabetes by assessing vascular function and endothelial phenotype before and after treatment with placebo or the anti-inflammatory drug salsalate, an inhibitor of NF:2 activation. We expect that these studies will improve our understanding of the pathogenesis of vascular disease in diabetes that will be relevant to the generation of innovative therapeutic and preventive therapies. PUBLIC HEALTH RELEVANCE: Diabetes is a critical and growing public health problem that affects over 170 million people worldwide and at least 20% of individuals over age 60. Cardiovascular complications of diabetes including myocardial infarction, heart failure, peripheral artery disease and stroke are responsible for more than 65% of all deaths in patients with diabetes. This applications aims to develop an improved understanding of the mechanisms of vascular injury in patients with diabetes and lead to the generation of new prevention and treatment strategies to reduce the burden of cardiovascular disease in this high risk group.